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  1. Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously address different antigens or epitopes. BsAbs with “two-target” functionality can interfere with multiple surface receptors or ligands associated. 1. Development history of three generations of bispecific antibodies The advent of hybridoma technology in the 1970s reminded people whether the same antibody could be used to target two different targets, namely Bispecific Antibody (bsAb). Most of the earliest bispecific antibodies are developed by chemical coupling methods. Although the method is much simple, the resulting products are complex and heterogeneous and difficult to use in industrial production. Until the 1980s, scFv-based recombinant bispecific antibodies appeared and gradually became the focus of research with the popularization of recombinant DNA technology. Amgen/Micromet’s BiTE (Bispecific T cell Engager) is the archetype of the first generation of bispecific antibodies, characterized by a simple structure and two scFvs connection. But it also has obvious disadvantages such as short half-life and low expression. A pump for continuous administration is clinically necessary due to the short half-life with only two hours. Fortunately, the clinical dose of BiTE is less than one tenth of the amount of common antibodies, which virtually solves the problem of mass production. The first recombinant bispecific antibody Blinatumomab (CD3-CD19 BiTE) was finally approved by the FDA in December 2014 for marketing in the United States after various difficulties. Genentech’s Paul Carter team invented the knob-into-holes bispecific antibody in the 1990s as the second-generation bispecific antibodies, with a structure and stability similar to that of natural IgG. Roche/Chugai’s Emicizumab (factor IX and factor X bsAb) using the knob-into-hole technology was approved by the FDA in November 2017 as the second bispecific antibody marketed in the United States. There are currently about 120 bispecific antibodies in different clinical development stages around the world, including third generation bispecific antibodies. If taking T cell-targeted therapy as an example, Roche’s CEA-TCB (CD3-CEA bispecific antibody) is a typical representative of third-generation bispecific antibodies, which utilizes the knob-into-hole technology of second-generation bispecific antibodies, and Roche engineers invented CrossMab technology shortly after the acquisition of Genentech and successfully solved the bottleneck of knob-into-hole in common light chain. In addition, CEA-TCB achieves bivalent binding of tumor antigens and monovalent binding of CD3, enabling the bispecific antibody to produce avidity effects when combined with tumor antigens while reducing CD3 antibody binding toxicity. At present, CEA-TCB has completed Phase 1 clinical trials and achieved satisfactory results. 2. The challenges for bispecific antibodies development Since 1986, the first antibody drug, Muromonab (OKT3), has been approved for marketing, the FDA has approved the listing of 73 antibody drugs by the end of 2017, and there are only two bispecific antibodies listed above. The development of bispecific antibodies lags significantly behind therapeutic monoclonal antibodies, which mainly results from the following reasons: Early bispecific antibody has a difficult expression and poor stability, as well as a complicated production process; The early development cost of bispecific antibodies is significantly higher than that of monoclonal antibodies; Bispecific antibody project involves target biology, structural biology, antibody engineering, and screening strategies. In 2017, the market capacity of antibody drugs broke through the $100 billion mark for the first time. Antibody drugs have become one of the fastest growing areas in the pharmaceutical industry. Bispecific antibodies at the forefront of antibody drug development have long been the development direction of major pharmaceutical companies around the world. It is believed that bispecific antibody will have a considerable market share in the field of solid tumor treatment. Compared with CAR-T, bispecific antibody drugs have the advantages of dose controllable and flexible drug administration period. At the same time, bispecific antibody drugs can also target other immune cells, such as NK, Macrophage, etc. Author Bio Creative Biolabs was established in 2004 by scientists who are dedicated to conquering of cancer. Over the past 10 years, Creative Biolabs has grown into a recognized world leader in antibody (rAb) discovery, engineering, production, and analysis. Standing on the shoulder of a giant, the bispecific antibody (BsAb) team has a collective of experienced scientists committed to providing high-quality services to customers all over the world. Now, with the cutting-edge platforms and methods (quadroma development, chemical conjugation, and genetic engineering), a comprehensive list of bispecific antibody products is available to customers in academia and industry fields.
  2. Brief Introduction to Short Tandem Repeats Microsatellite DNA, also known as short tandem repeats (STR) or simple repeat sequences (SRS or SSR), is widely found in prokaryotic and eukaryotic genomes, consisting of a unit of two to thirteen nucleotides repeated hundreds of times in a row on the DNA strand which is about 5% of the eukaryotic genome, the basic unit (core sequence) is 1-6bp. The most common of these is (CA) n and (TG) n, and the human genome has about 5 × 104 ~ 1 × 105 (CA) n repeats which take 10% of the genome. Each microsatellite DNA has the same core sequence structure, the number of repeating units is about 10 to 60 times, and its length is generally not more than 300bp, mostly located in the non-coding region of the gene, intron or untranslated region. which may be present in the Alu sequence or Satellite sequence, but in the coding sequence and exon also can find the presence of microsatellite DNA. The high polymorphism of microsatellite DNA is mainly due to the difference in the number of tandem numbers. There is a big difference in the distribution for microsatellite DNA in different races and populations due to the number of repeat units and repetition, which constituted STR genetic polymorphism. And the number of repetitions between different individuals at a homologous STR site is also different so that STR loci analysis can identify individuals that are similar to fingerprint recognition. It is possible to create a personal gene file by identifying a specific sequence of genomes at particular loci. Currently, there are more than 10,000 STR loci are available. STR analysis has become an important analytical method for individual identification and paternity testing in the field of forensic science. It can be applied to judicial case investigation, that is, genetic fingerprint analysis. The Causes of STR The replication slip caused by mismatches between DNA strands during the mitotic process is considered to be the most common cause of the occurrence of STR, and in general, there will be an average of one-thousandths of microsatellite DNA will undergo replication slippage. The study showed that the rate of tandem duplication at repeat sequences was higher than the probability of point mutations occurring elsewhere in the genome. Most of the replication slides only cause a change in the repeat unit, and the probability of replication slip is different due to the size of the different copy units and different species. STR Detection Method STR analysis is one of the most useful methods in molecular biology which is used to compare specific loci on DNA from two or more samples. There are two common methods for STR detection: capillary electrophoresis (CE) and gel electrophoresis, which can be used to determine the specific amount of microbes Satellite sequence and draw the STR map. Typically, each allele is shared by about 5-20% of people. And the advantages of STR analysis will be reflected in the simultaneous identification of multiple STR loci. Each individual can be identified accurately by the resulting STR map. In theory, if there were 16 STR loci being used in combination, the recognition rate will be 0.999999999998. The Importance of STR Analysis It is still common for cell lines to be misidentified and cross-contamination, although scientists use a variety of traditional methods to identify cells, there are still dozens of cross-contamination happened. And even some researchers found that cell lines were misidentified or cross-contamination while cell identification reports for the higher score study articles publication, resulting in erroneous conclusions. All of which will lead to the waste of research funding and time, and resulting in a large number of invalid or erroneous data that will mislead other researchers. Based on the statistics data, around 20% of the cell lines were misidentified and cross-contamination in the labs so that it is a serious concern for researchers to provide accurate cell line identification and prevent cell lines from cross-contamination. Currently, several STR loci have been developed to analyze cross-contamination and cell types at the same time, that can detect up to 0.1 ng of DNA (about 15 Diploid genomes) with high sensitivity for the trace pollution. All the mentioned above is the complete guide for the Short Tandem Repeat and its importance!
  3. Firstly, let us see two antibodies: 4G2 antibody This product is a mouse monoclonal antibody that specifically recognizes E protein from DENV-2. The E protein-binding antibody 4G2 is an epitope-specific antibody that can be used in ELISA. A11 antibody Recombinant Human Antibody (EDE2 A11) is capable of binding to DENV E proteins, expressed in HEK 293 cells as the combination of a heavy chain (HC) containing VH from anti-DENV E proteins mAb and CH1-3 region of human IgG1 and a light chain (LC) encoding VL from anti-DENV E proteins mAb and CL of human kappa light chain. So, what is DENV? Figure1. Dengue virus Dengue virus (DENV) belongs to a serotype subgroup of flaviviridae. Its morphological structure is similar to that of Japanese encephalitis virus (JEV), but its size is small, about 17-25 nm. It can be divided into four serotypes according to antigenicity, 1, 2, 3 and 4 serotypes. There are antigenic differences among different viral strains in the same type. Viral characteristics and physicochemical properties DENV has a capsule and is a single positive strand RNA virus without segments. The viral genome is about 11 KB long. It encodes three structural proteins (C-prM-E) and seven non-structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). Among them, E protein is the main structural protein of the virus, which determines the histophilicity of the virus and mediates the binding of the virus to cell receptors. At the same time, it is also an important component of influencing virulence, causing protective immune response and immunopathological damage. The virus is sensitive to heat and can be inactivated at 56 ℃ for 30 minutes. Lipid solvents such as chloroform, acetone, lipase or sodium deoxycholate can inactivate the virus by destroying its envelope. Viruses are sensitive to gastric acid, bile and protease, and to ultraviolet and gamma rays. Antigenicity and serotype According to the antigenicity of virus E protein, it can be divided into four serotypes: DENV1, DENV2, DENV3 and DENV4. The antigenicity of these viruses was cross-linked, but not with other antigens of flaviviridae. Antigenic determinants of viral envelope protein E can induce protective neutralizing antibodies and hemagglutination inhibitory antibodies, and may be involved in the occurrence of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). NS1 and NS3 are both immunoreactive and immunogenic, which can induce protective immunity against dengue virus homology in mice. Clinical symptoms Dengue virus often causes asymptomatic recessive infection. The main clinical manifestations of the patients are dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The main clinical manifestations of DF include protruding fever, severe headache (mostly frontal pain), post-ocular pain (aggravation of eyeball movement), general pain and joint pain, nausea and vomiting, lymphadenopathy, leukopenia and thrombocytopenia. DF not only causes heart, liver, lung, kidney, brain and other system damage, but also leads to myocardial damage. The clinical manifestations of DHF/DSS are more serious. In addition to the above DF symptoms, severe and persistent abdominal pain, nasal, oral and gingival bleeding, skin bleeding and ecchymosis, black stool, extreme thirst, pale skin and mucosa, chills, restlessness or sleepiness, hematocrit > 35% and other manifestations can occur. Serious cases can cause death. DENV infection can also cause encephalitis. Pathogenesis The pathogenesis of dengue fever and dengue haemorrhagic fever has not been fully elucidated so far. It is generally believed that the complex interaction between virus and host causes severe risk factors, including patient's age, virus serotype and genotype, and host's genetic background. Recent studies have shown that there are several hypotheses about the pathogenesis: viral affinity, viral toxicology, host susceptibility, antibody-dependent enhancement (ADE), cross-reactive T cell response and cytokine storm. This paper briefly introduces ADE and cytokine storm. Scholars believe that there are group-specific determinants and type-specific determinants on the surface of DENV. The antibodies produced by the former have a strong enhancement effect on DENV infection, called enhanced antibodies, while the antibodies produced by the latter are called neutralizing antibodies. When the body is re-infected with different types of DENV, serum neutralizing antibodies cannot completely neutralize the virus. Enhanced antibodies can bind with the virus to form immune complexes. These immune complexes promote the replication of the virus in these cells through Fc receptors on the membrane of monocytes or macrophages, resulting in ADE effect, leading to clinical outcomes in patients. Symptoms worsen, blood concentration and shock occur, resulting in DHF/DSS. However, DHF/DSS can also occur in many patients with primary infection, indicating that there are other pathogenic factors. Cytokines are small molecular weight soluble proteins or polypeptides secreted by immune cells and other types of cells. They have the functions of transmitting information between cells, regulating immune response, and participating in inflammatory damage and other pathological processes. However, excessive inflammation can cause systemic inflammatory response syndrome and make the body in a state of hypermetabolism and hyperdynamic circulation. Over-release of inflammatory cytokines may lead to multiple organ dysfunction syndrome and even death. The control of inflammation is essential in the treatment of many diseases. It was found that the levels of IL-1, IL-4, IL-6, IL-13 and TNF-alpha were significantly increased in severe patients with dengue fever. There is a significant correlation between TNF-alpha and thrombocytopenia. Studies have shown that the transition from Th1-type response to Th2-type response is an important mechanism of DHF/DSS. Cytokines in severe patients are mostly Th2-type cytokines. After virus infection, excessive release of cytokines and inflammatory factors increases vascular permeability, leading to hemorrhage and shock. Diagnosis The diagnosis of dengue virus is mainly a combination of clinical symptoms and laboratory diagnosis. Laboratory diagnosis of dengue virus includes virus isolation, antigen and antibody detection, gene detection and so on. Virus isolation Virus isolation is the golden standard for the diagnosis of viral diseases. The duration of dengue viremia is relatively short. Samples should be collected within 4 to 5 days of the onset of fever. Virus isolation and culture can be carried out by living mosquitoes, cells or animals. Antigen detection NS1 protein is the main marker antigen of dengue virus infection. NS1 protein appears earlier than IgM antibody and can be used for early diagnosis. At present, several commercial kits have been used to detect the NS1 antigen of dengue virus. Serological detection The serological method is more convenient because the humoral immune response of patients infected with dengue virus lasts for at least several weeks after the onset of the disease and the sample can be collected for a relatively long time. Detection of IgM, IgG capture ELISA, IgM, IgG indirect ELISA and hemagglutination inhibition test are the most commonly used serological methods for diagnosis of dengue virus infection. Gene diagnosis In recent years, the molecular biology diagnostic methods of dengue virus have been enhanced. Nucleic acid amplification has become the most important method for rapid diagnosis of dengue virus. Various RT-PCR methods try to detect the RNA of dengue virus. The nucleic acid amplification technique can be used to identify the serotype of dengue virus as well as to detect the virus. Owing to its high throughput and parallel processing, gene chip technology can detect and analyze pathogens comprehensively. Its simple and fast operation, low cost and small size provide wide application prospects in the fields of molecular biology, disease diagnosis and treatment. Scientists used PCR to amplify the nearly full-length dengue virus 2 gene, digested the amplified products by enzyme-digestion-PCR, and constructed the dengue virus 2 DNA library to obtain microarray probes. The probe was fabricated into a DNA chip for detection of type 2 dengue virus by dot analyzer. The results showed that the hybridization of the sample with type 2 dengue virus gene chip was highly sensitive and specific. Dengue fever, as an important vector-borne infectious disease, has been increasing year by year, seriously endangering human health. Although some achievements have been made in the related research, there are still many problems to be solved urgently. There is still no consensus on the pathogenesis of dengue fever, especially in severe cases. There is no effective prevention and treatment measures. Future work should start with the mechanism of virus and host, obtain effective treatment of dengue fever as soon as possible, and todevelop a protective multivalent vaccine.
  4. August 16, 2019, as a leading supplier and manufacturer in antibody field, Creative Biolabs recently has updated its new product types for therapeutic antibody, which has at least three types of mechanisms. For instance, the neutralizing effect of the therapeutic antibody on its ligand directly binds its ligand, so that its ligand does not act on the target tissue and cells to trigger an undesired reaction; antibody-mediated cytotoxicity of the antibody binds to the target cell, Cytotoxicity that triggers immune cells; complement-mediated cytotoxicity antibodies have a complement-binding site that, upon binding of the antibody to the target cell, triggers the complement effect and kills the target cell. These new items of Creative Biolabs are including but not limited to: Anti-CD34 Therapeutic Antibody Anti-CD55 Therapeutic Antibody Anti-CD81 Therapeutic Antibody ... “those newly-updated products in therapeutic antibodies will bring a lot to our customers in their research programs. Our experienced scientists can guarantee the high quality of them and they can perform well during all kind of research items to assist researchers to achieve success. The anti-34 therapeutic antibody is a kind of recombinant humanized monoclonal antibody expressed in CHO binding to human CD34. It can recognize the human cd34 antibody membrane-protein in the cell membrane and perform well in the clinical treatment of hematologic malignancies and solid tumors, etc. The anti-CD55 therapeutic antibody is binding to human CD55. cd55 antibody is built with strong potential to be applied as an additive agent to treat gastric cancers. Studies show that a single 20mg dose can induce the cell killing in human primary gastric tumors, which results to tumour cell regression in over half of PAT-SC1-treated patients. And we do believe patients with gastric cancers can be totally free in the nearly future. The target of anti cd81 antibody is human CD81, which is generated by genetic immunization. It is very promising in the virus-host interactions. All in all, newly-updated products will bring Creative Biolabs another shine.” Dr. Carlose Smith explained, a senior scientist in R&D of Creative Biolabs. “with the faith of providing high-quality products and professional services, Creative Biolabs is always trying its best to develop products and services needed by our customers. And we do offer the custom services and products for some specified items. Of course, Creative Biolabs has gained a good fame among our customers and other biological companies.”Dr. Carlose added. We do believe that Creative Biolabs will be much more better in the near future due to its dutiful spirit of enterprise. Now, it has been well received among customers. About Creative Biolabs With more than ten-year corporate history, Creative Biolabs has accommodated many excellent researchers at home and abroad to develop high-quality and professional products and services belonging to the antibody field, which is now getting more and more popular and needed by clinical researches. The future for Creative Biolabs is getting more and more promising due to its professional scientists and research staff.
  5. Relapsing fever is transmitted via arthropod vectors. The human body louse (Pediculus humanus corporis) transmits Borrelia recurrentis, the causative organism of louse-borne relapsing fever (LBRF), while Ornithodoros ticks are vectors for the at least 15 different species of Borrelia that cause endemic or tick-borne relapsing fever (TBRF). While humans are the only known host for the LBRF spirochete, the TBRF-causing Borrelia species (with the exception of B. duttonii) have their reservoirs in small rodents and have been found in several locations worldwide. In North America, three species of Borrelia cause TBRF, and several outbreaks of the disease have been described. In East Africa, B. duttonii is the principal cause of TBRF. Antibody responses against relapsing fever Borrelia species are primarily directed against outer surface lipoproteins. Two major protein groups have been identified, namely, variable small proteins (approximately 22 kDa) and variable large proteins (approximately 38 kDa). These proteins have been studied most thoroughly for B. hermsii and B. turicatae. It is probable that all of the relapsing fever Borrelia species share the same antigenic variation scheme as that described in detail for these two species. When spirochetes are present in blood, they must evade the immune defense systems. Before the acquired immune responses lead to the production of antibodies, the alternative pathway of complement operates as a major innate immune defense system against the invading organisms. In the presence of antibodies, complement acts as an effector system, mainly via the classical pathway (CP). Both pathways lead to coating of the target surface with C3b. Together with their cleavage products, such as iC3b, the C3b molecules opsonize the target for phagocytosis. Further activation can lead to the formation of lytic membrane attack complexes. To avoid overconsumption of the components of the complement cascade and to protect self-cells from harmful attacks, complement activation must be tightly regulated. This is mediated by regulatory proteins in plasma and on cell surfaces. The major fluid-phase regulators of complement are factor H (FH), for antibody-independent alternative pathway activation, and C4b-binding protein (C4BP), for antibody-dependent CP activation. These regulators accelerate decay of the C3 convertases (C3bBb and C4b2a, respectively) and act as cofactors for the irreversible inactivation of C3b and C4b, respectively. As a net effect, these functions prevent complement-mediated destruction of the target in both the absence and presence of antibodies. Acquisition of the host plasma complement regulator FH has been shown to be beneficial for complement evasion among other spirochetes, such as Borrelia burgdorferi sensu stricto and B. afzelii, which express at least two FH binding proteins. Also, the relapsing fever agents B. hermsii and B. parkeriihave been shown to bind FH, while no binding has been observed for B. turicatae. B. hermsiiexpresses a unique 20kDa outer surface protein (FhbA) responsible for FH binding. The bacteria that cause human Lyme disease belong to a group of at least 15 species, referred to as Borrelia burgdorferi sensu lato, or the Lyme disease agent bacterial group. Among these, B.burgdorferi sensu stricto causes Lyme disease in North America, while in Europe and eastern Asia Borrelia afzelii, Borrelia garinii, and Borrelia bavariensis sp. nov. are the best-known causes. However, more recently, Borrelia bissettii, Borrelia lusitaniae, Borrelia spielmanii, and Borrelia valaisiana have been isolated from Lyme disease patients. Other species in this bacterial group, such as Borrelia japonica and Borrelia sincia in Asia, have not been associated with human disease. To date, genome sequences have been reported for 14 B. burgdorferi isolates, two B. afzelii isolates, two B. garinii isolates, one B. bavariensis sp. nov. isolate and 1 isolate of unassigned species. The complete genome sequences for three additional Borrelia species: B. valaisiana isolate VS116, B. bissettii isolate DN127 clone 9, and B. spielmanii isolate A14S. DNA samples from low-passage isolates were sequenced to minimize plasmid loss, and genomes are sequenced to about 8-fold coverage as previously described. Genome annotation is performed using the JCVI Prokaryotic Annotation Pipeline . The DN127 chromosome and 35 of 39 plasmid sequence contigs are closed, but in order to maximize the use of available funds, the sequences of a few replicons are not closed and some gaps remained in these sequences (two chromosomes and one cp9 and three cp32 plasmids, because they are much less variable than the other plasmids). These three genome sequences include 3,914,891 bp in total (1,258,865, 1,403,466, and 1,252,560 bp for strains VS116, DN127, and A14S, respectively), with an average of 1,304,497 bp/genome. Like the sequences of other Borrelia species, they include numerous linear plasmids (6, 7, and 7, respectively) and circular plasmids (2, 2, and 2, respectively). Plasmid numbers in these three strains range from 11 in VS116 and 12 in A14S to 16 in DN127. Plasmids that are very similar to B. burgdorferi sensu stricto cp26, cp32 (7 in DN127, versus 3 in the other two strains analyzed), and lp54 plasmids are present in each of these isolates, and DN127 also contains an unusual fusion of four partial cp32 plasmids. Plasmids with predicted lp17 compatibility are also present in all three genomes, making it the only other plasmid type, in addition to cp26 and lp54, to be found in all 23 B. burgdorferi sensu lato sequenced genomes. However, the gene contents of the lp17s are much more variable than the other universally present plasmids. The detailed analyses of these genome sequences will be a major step forward in attaining a complete understanding of B. burgdorferi sensu lato diversity. They will contribute to the development of species- and group-specific vaccines and diagnostic tools, as well as inform us whether these species are in genetic contact with the more-common Lyme disease-associated agents. Reference [1] Barbour A G, Hayes S F. Biology of Borrelia species. [J]. Microbiological Reviews, 1986, 50(4):381-400. [2] Van H C, Comberbach M, De G D, et al. Evaluation of the safety, reactogenicity and immunogenicity of three recombinant outer surface protein (OspA) lyme vaccines in healthy adults [J]. Vaccine, 1996, 14(17–18):1620-1626. [3] Kraiczy P. Whole-genome sequences of Borrelia bissettii, Borrelia valaisiana, and Borrelia spielmanii. [J]. Journal of Bacteriology, 2012, 194(2):545-6. [4] Cutler S J, Moss J, Fukunaga M, et al. Borrelia recurrentis characterization and comparison with relapsing-fever, Lyme-associated, and other Borrelia spp.[J]. International Journal of Systematic Bacteriology, 1997, 47(4):958-68.
  6. Antibody drugs are one of the fastest growing biopharmaceuticals, which bring huge benefits to the innovative drug market, with anti-tumor antibody drugs predominating. Since rituximab, the first antibody drug for cancer treatment, was approved by the US FDA in 1979, 17 antibody drugs have been approved for cancer treatment, but the gemtuzumab has withdrawn from the market. According to statistics from 2013, there are about 350 antibody drugs currently in clinical research, most of which are in the early evaluation stage. Therapeutic antibodies in Phase III clinical studies include 28 monoclonal antibodies and a mixture of monoclonal antibodies mainly used for cancer, inflammation or immune diseases, Alzheimer's disease and infectious diseases, 10 of which are cancer therapeutic antibodies.There are also many antibody drugs in preclinical studies, and their research trends are mainly focused on the following aspects, including: the discovery and confirmation of new antibody targets, new strategies to reduce antibody immunogenicity, conjugates of antibodies and drugs, and research on bispecific antibodies as well as other novel antibody drugs. 1.Discover new antibody target molecules At present, the target molecules of antibody drugs are mainly divided into three categories: 1. Clinically validated target molecules, the efficacy of which has been confirmed, for example: CD20, HER2, EpCAM and CTLA-4; 2. Experimentally verified target molecules, the efficacy of this type of target molecule has been confirmed in cells and animals, such as IGF-1R, CD19, CD80, CXCR4 and ICAM-1; 3. New functional target molecules, newly discovered target molecules with certain efficacy, such as RAAG12, CD151, TSN-1, etc. To date, only a few target molecules for anti-tumor antibody drugs have been approved for marketing, and currently only nine. Many tumor-associated biomarker molecules have been reported, and thus so many target molecules for antibody therapy can be excavated. Even if the same target molecule has been developed, different molecular epitopes exist, and antibody drugs against different epitopes can be developed. For example, 5. Anti-CD20 antibody drugs have been marketed, and the 5 are now in clinical research. In addition, many tumor cell transmembrane receptors and extracellular matrix-related genes can be spliced by differential splicing, which can generate new editing sequences and form new differential receptors and substrates as new antibody target molecules. The differentially spliced gene variants have been reported to be: FGFR variants, EGFR variants, EpCAM variants, L1CAM variants, Claudin18 and CD44 variants, and splicing of versican VN, fibronectin F and prion protein T Variants, etc. In recent years, many tumor-associated antibody target molecules have been studied, including EpCAM, PSMA and folate-binding proteins, as well as gangliosides, αVβ3, TRAILR2, FAP and toughlin. Recently, glypican proteoglycan has also been reported as a new target molecule for the treatment of liver cancer antibodies. Reduce antibody immunogenicity Many antibody drugs are murine antibodies, which produce human anti-mouse antibody (HAMA) responses in clinical treatment. Therefore, for long-term treatment, an antibody drug that requires repeated administration is likely to produce a HAMA reaction, which requires humanization to reduce its immunogenicity. Humanized antibodies include chimeric antibodies, modified antibodies, and the like, which have biological properties such as reduced HAMA response and prolonged half-life in blood. In contrast to murine antibodies, the functional effector portion of the humanized antibody drug can be engineered as needed, and the humanized constant region of the antibody prevents the production of anti-isotype antibodies during treatment. The modified antibody further reduces the proportion of the murine fraction in the antibody compared to the chimeric antibody, and significantly reduces the HAMA response. In addition, the study of fully human antibodies has also received great attention and is one of the important development directions of antibody drug research. Fully human antibodies can be obtained by phage display technology, antibody library screening technology and transgenic animal technology. The anti-tumor antibody drugs produced by this technology have obvious inhibitory effects on cancer growth. The immunogenicity of antibody drugs is one of its major toxic side effects, but there is no reliable prediction method for the frequency and timing of immunogenicity and antibody aggregation. If these problems are solved, new antibody detection techniques need to be developed. The results of studying the immunogenicity of human-derived antibodies, humanized antibodies and murine antibodies in humans indicate that: 1. More than 20% of mouse-derived antibodies induce tolerable immunogenicity, or negligible. These mouse antibodies do not need to be humanized; 2. Humanized antibodies can reduce immunogenicity, but their clinical therapeutic effects are also significantly reduced; 3. The immunogenicity of antibodies from different sources is not completely consistent with the law. A small number of humanized antibodies and fully human antibodies also produce significant immune responses; 4. Not only do we need to evaluate the utility of fully human and humanized antibodies item by item, but we also need to consider cost-effectiveness, while considering biochemical characteristics and targeted treatment indications. To date, there is no good way to completely eliminate the risk of antibodies binding to host cells. But classical research suggests that monomeric immunoglobulins are inherently tolerable if we are able to create methods to prevent antibody aggregation or immune complex formation. To be continued in Section Two…
  7. Antibody Engineering comprises in vitro selection and modification of human antibodies, including humanization of mouse antibodies for therapy, diagnosis, and research. It holds the potential for creating antibodies with multiple specificities, greater affinity for their targets, and fewer side effects. In this article, the development history and types of antibody engineering drugs are talked. Antibodies as therapeutic agents An antibody is an immunoglobulin molecule with a specific amino acid sequence induced by an antigen, synthesized and secreted by lymphocytes (plasma cells). An antibody can bind to an antigen such as a bacterium, a virus or a toxin, and neutralize and remove the harmful substance by the following three or one of the following methods: a. directly inactivates the antigen; b. the immune effector cells phagocytose and destroy it; c. it is weakened by the surface of the antigen and thus be easily damaged by complement. The study of antibodies as therapeutic agents has been going on for a century. The lateral chain theory proposed by Ehrlich laid the foundation for immunology and immunotherapy. He believes that the surface of the cell has specific receptor molecules (or side chains) that bind only to specific groups in the toxin molecule; if the cells survive with the toxin, they will produce excess side chains. Some of the side chains are released into the blood, which is an anti-toxin. This is what is now called an antibody. Ehrlich uses organic arsenic compounds to treat syphilis and introduces the term "magic bullet" in the field of chemotherapy to strengthen the strategies and goals of specific therapies. As early as 1895, it was reported that Hericourt and Richet injected cancer cells into animals to produce antiserum for the treatment of cancer patients, and the symptoms of the patients were significantly improved. In the early 20th century, many researchers repeated the above tests, but failed to obtain a definite therapeutic effect, and sometimes even produced serious side effects. Later studies have found that antisera contain a large number of different kinds of antibodies, which are directed against different antigens on the surface of cancer cells, and these antigens are also present in normal tissue cells. Therefore, the cross-reactivity of antibodies with normal tissues may lead to serious consequences. Antiserum has long been effective in neutralizing exogenous toxins such as snake venom, but has not been effective in the treatment of tumors and other diseases. Monoclonal antibody In 1975, Köhler and Milstein used B lymphocyte hybridoma technology to prepare monoclonal antibodies (MAb). Monoclonal antibodies have high specificity, uniform properties and are easy to mass produce. A variety of monoclonal antibodies can be prepared in vitro by cell engineering, which is an epoch-making advance in antibody production. It is especially important that monoclonal antibodies have high specificity for the corresponding antigens and homogeneity of the antibody molecules, which can greatly reduce cross-reactivity with normal tissues in vivo, and bring new hope for the treatment of diseases by using antibodies, especially for treating tumors. At that time, some people called it "magic bullet", and it is expected that monoclonal antibodies can specifically attack pathogenic cells or pathogens without causing toxic side effects. In the past 20 years, monoclonal antibodies have been widely used in the diagnosis of diseases, and make much breakthroughs have despite many obstacles. To distinguish, an antibody previously prepared by immunizing an animal with an antiserum pathway is referred to as a polyclonal antibody, or a conventional antibody. Genetically engineered antibodies Since the monoclonal antibodies prepared by the B lymphocyte hybridoma technique are mostly murine, the human anti-mouse antibody (HAMA) reaction can be induced in the human body, which limits the application of the monoclonal antibody as a therapeutic agent in humans. In order to overcome the heterologous reaction of murine monoclonal antibodies, in the mid-1980s, researchers explored the genetic engineering of mouse-derived monoclonal antibodies to prepare humanized antibodies. For example, they spliced the variable region of mouse Ig to the constant region of human Ig or modified the CDR region of the murine Ig to the human Ig. At the same time, considering the treatment of solid tumors with intact antibodies, the molecules are too large to penetrate the extracellular space into the deep tumor, so genetic engineering methods are used to miniaturize antibody molecules, such as single-chain antibodies (scFv), diabody, minibody, etc. The establishment and development of antibody library technology can directly clone the gene of specific antibody in the prokaryotic cell system by genetic engineering method, which greatly promotes the preparation and research and development of novel genetic engineering antibodies. Antibody engineering has become a key technology to promote the development of antibody drugs. Immunoconjugates and fusion proteins The molecular structure and function of the antibody has two sides. One is the specific binding to the antigen and is completed by the variable region. The second is the effect function triggered by binding to the antigen, which is completed by the constant region. In order to enhance the effector function of antibody drugs, especially tumor antibody drugs, and to enhance their killing effect on tumor cells, an effector molecule or a "warhead" substance is commonly used to chemically connect with an antibody to prepare an immunoconjugate. The specificity of the antibody is utilized as a targeting vector; the "target" is used to kill tumor target cells. There are three main types of substances used as "warheads", namely radionuclides, chemotherapeutics and toxins. These "warhead" substances are linked to antibodies, which constitute radioimmunoconjugates, chemical immunoconjugates and immunotoxins, respectively. A fusion protein can be produced by a genetic engineering method, and the antibody portion of the fusion protein is generally a single-chain antibody scFv; the portion corresponding to the "warhead" is generally a toxin fragment, a cytokine or a peptide drug. The monoclonal antibodies, antibody fragments, genetically engineered antibodies, and immunoconjugates described above can be collectively referred to as antibody-based drugs, also known as monoclonal antibody therapeutics. Such antibody drugs are prepared by a cell engineering method or a genetic engineering method or a combination of two methods, and may also be referred to as an antibody engineering drug. Antibody drugs for clinical application The development of antibody drugs has a prominent position in the field of biotechnology drugs. The number of antibody drugs currently in preclinical and clinical research stages is among the highest in biotechnology drugs. The variety of antibody drugs in research and development is versatile, and the diseases that may be used for treatment include cancer, viruses and other infectious diseases, cardiovascular diseases and immune system diseases, among which cancer treatment is the first.
  8. Bridging cells (in-transbinding)—bridging cell method The most commonly used bridging cells are bridging effector T cells and tumor cells, also known as T cell-engaging bsAb (bsTCE). In addition, NK cells, stem cells, and tumor cells etc. are also used. bsTCE directly couples T cells and tumor cells to form immune synapses, activates in TCR, releases granzymes and perforin, and finally lyses tumor cells. BsTCEs bind to CD3ε in the CD3ε-TCR complex and activate T cells without antigen presentation. Although this mechanism was proven to be highly effective in the laboratory by co-culture experiments, it caused rapid uncontrolled cytokine release syndrome (CRS), including the earliest marketed CD3×EpCAM bsTCE catumaxomab, via the Fc segment binding to FCγR expressing Kupffer cells in the liver and causes uncontrolled hepatotoxicity. Targeting bsTCEs by CD3 requires complete inhibition of Fc-mediated effector function to reduce non-target toxicity and maximize therapeutic effects. Tumor target—Target expression and efficacy The activity of some bsTCE is positively correlated with the expression level of tumor targets, such as CEA, CD33, HER2. There are also some no significant correlations with expression levels, such as EPHA2, PSMA. However, there is also a minimum required tumor target expression, such as RG6106 requires at least 50 bsTCE binds to the myeloma surface FCRL5, while Roche cibisatamab requires 10,000 CEA in combination with bsTCE to be activated. Structural design Bivalent tumor targets can increase drug binding activity and enhance drug efficacy. Some tetravalent bsTCEs are under development, including Adaptir and TandAb, which have two CD3 and two TAA binding units. In addition, trivalent bsTCEs are under development, in which CD3 is monovalent and TAA is bivalent. For example, RG6026 (also known as RO7082859; Roche). Immune checkpoint Clinically administration of monoclonal antibody against CTLA-4, PD1/PD-L1 immunological checkpoints have been quite successful. More than 10 corresponding bispecific antibodies have entered the early clinical stage worldwide, involving PD1/PD-L, CTLA4, lymphocyte activation Gene 3 (LAG3) or T cell immunoglobulin mucin protein 3 (TIM3; also known as HAVCR2) Previously, two checkpoint antibodies were combined to improve efficacy. For example, treatment of melanoma patients with ipilimumab (anti-CTLA4) + nivolumab (anti-PD1) improves survival compared with ipilimumab alone. However, clinical immune-related side effects have also multiplied. In order to improve the safety of the combined targeting of PD-1 and CTLA 4, an Fc-silencing bsAb, a high affinity inhibition of PD-1, and a low affinity binding inhibition of CTLA 4 were designed to inhibit the PD-1 pathway. This design is beneficial for inhibiting PD-1-CTLA 4 double positive tumor infiltrating lymphocytes, and at the same time reducing the binding of CTLA 4 expressing peripheral T cells, thereby providing better safety. Currently, the safety and early efficacy of the four PD-1×CTLA4 bsAbs are being evaluated in early clinical trials. The concept of blocking two immunological checkpoint inhibitors is also clinically used for other target combinations, such as PD-1 x LAG 3, PD-1 x TIM3, and PD-L1 x CTLA 4. Bridging receptors (in-cisbinding)—bridge acceptor inhibitory activity Targeting inhibition of tumor receptor tyrosine kinases (RTKs) such as EGFR and HER 2 is a successful anticancer approach, but the development of drug resistance is one of the major limiting factors for such treatment. Drug resistance typically involves up-regulation of other RTKs that bypass specific receptor inhibition to activate parallel signaling pathways. For example, in the tyrosine protein kinase MET pathway, non-small cell lung cancer (NSCLC) tumors are resistant to EGFR tyrosine kinase inhibitors. JNJ-61186372 (Janssen Pharmaceuticals) is an EGFR x MET bsA that blocks EGFR and MET signaling by inhibiting ligand-induced activation and receptor degradation by cFAE. JNJ-61186372 has antibody-dependent cytotoxic activity (ADCC) which is produced by Fc containing low fucose. The glycotypic analysis in the quality control of such bispecific antibodies should be noted. Bridging receptors (in-cisbinding)—bridge acceptor agonist receptor activity In contrast to blocking pathogenic signals with inhibitory antibodies, some therapeutic concepts require receptor signals to be activated by agonistic antibodies. Specific bsAbs are also particularly suitable for activating multi-component receptor complexes because they require simultaneous binding of the receptor and the co-receptor to activate. Activation of the fibroblast growth factor 21 (FGF 21) pathway has been reported to improve obesity and diabetes. However, the pharmacokinetic properties of recombinant FGF21 are poor, and chronic treatment has a risk of adverse reactions. Thus, the agonist bFKB8488A (Roche) was designed to activate this metabolic pathway by selectively targeting the fibroblast growth factor receptor 1C (FGFR1C)-β-Klotho (KLB) receptor complex. KLB is selectively expressed in liver, fat, and pancreatic tissues, while FGFR1C is widely distributed in liver, fat, and pancreatic tissues. Thus, co-targeting of these receptors may limit signal activation to only tissues that co-express KLB and FGFR1C and limit the adverse consequences of extensive FGFR activation, such as induction of cell proliferation. Preliminary results from an ongoing human first trial showed an improvement in cardiac metabolism in obese patients with insulin resistance. Cofactor mimetics Sampi and his colleagues began producing a bsAb to replace FVIII as a potential treatment for hemophilia A to prevent bleeding from FVIII dysfunction. The role of bsAb is to mimic the activated form of FVIII, FVIIIa, which binds FX and FIXa to enhance the catalytic activity of FIXa. In a wide range of screening efforts, a dual-characteristic antibody called AE 910 or emicizumab was finally selected and finally approved by the FDA for routine prevention to reduce bleeding in patients with hemophilia A using FVIII inhibitors. Further approval in October 2018 also included the use of emicizumab for prevention in patients without FVIII inhibitors. Emicizumab was first approved in Europe in March 2018. Piggyback approaches The first target-specific transport of a second specific target using the bsAb is referred to as a "piggyback transport" or "hijacking" method for accessing a restricted cell chamber. Raso and colleagues described the first case of the use of hijacking concept-specific transport proteins, which linked bispecific receptors to ricin A, mediating toxin internalization and toxicity. A recent example is the hijacking of the transferrin transport pathway to cross the blood-brain barrier and enter the immune-immunized brain region. The researchers targeted the transferrin receptor (Tfr) by using a binding arm of the bsAb, and the second binding arm carries β-secretase 1 (BACE 1), which transports BACE1 into the brain, and TfR×BACE 1 bsAb can reduce amyloid (Aβ, Aβ) peptide levels in brain tissue and cerebrospinal fluid. Psl×pcrvbsAb MEDI3902 is a full-length IgG 1 antibody inserted between Fabs and fc to form a symmetrical 2+2 format. This antibody enhances P. aeruginosa killing effect by neutrophils using a similar piggyback mechanism. Reference Oberst, .mAbs 6, 1571–1584 (2014). Laszlo, GS et al. Blood 123, 554–561 (2014). Lopez-Albaitero, A. et al. . Oncoimmunology 6, e1267891 (2017) Wolchok, J. D. et al. N.Engl. J. Med. 377, 1345–1356 (2017). Dovedi, S. J. et al. Cancer Res. 78,2776(2018). Hedvat, M. et al.Presented at the 2018 Society for ImmunotherapyofCancer (SITC) (2018). Xu, J. et al. . Am. J. Physiol. Endocrinol. Metab. 297,E1105–E1114(2009).Wu, A. L. et al.Sci. Transl Med. 3, 113ra126 Xu, J. et al. . Am. J. Physiol. Endocrinol. Metab. 297, E1105–E1114 (2009). Wu, A. L. et al. Sci. Transl Med. 3, 113ra126 Sampei, Z. et al. PLOS ONE 8, e57479 (2013)
  9. Anti-drug conjugate (ADC) is an antibody that binds to cytotoxic drugs and targets cytotoxic drugs to tumors through the targeting of antibodies, thereby reducing the non-specific systemic toxicity of drugs commonly found in chemotherapy. The study of antibody-drug conjugate (ADC) can date back to 1980s. A successful antibody drug conjugate drug includes four main parts: a suitable target (tumor antigen), a highly specific antibody, an ideal linker, and a highly cytotoxic drug. Firgure. The model of ADC action The basis for target selection Nowadays, ADC drugs are mainly used for anti-tumor. When selecting a target, the ideal target antigen should be overexpressed on the surface of tumor cells, but no expression or very low expression in normal tissues. Moreover, when the antibody and the target are aggregated in the ADC drug, it can be effectively internalized, and the drug is released into the cell to kill the target cell. Antibody specificity, affinity, and pharmacokinetics The high affinity of the antibody and the target antigen is the core of the effective targeting of the ADC. It is generally believed that the affinity index KD 10 nM is a basic requirement for the antibody. On the basis of this, antibodies that are low in immunogenicity, long in half-life, and stable in blood are screened. Selection and intracellular drug release The ideal linker can maintain stability in the blood and effectively release the drug in the target cells. The commonly used Linkers can be divided into two categories: cleavable linkers and non-cleavable linkers. The current study found that seven B cell receptors (CD19, CD20, CD21, CD22, CD79b, and CD180) have effective effects by cleavable linkers. In contrast, when non-cleavable linkers are used, only CD22 and CD79b antigens can bind to the antibody, effectively transport ADC to lysosomes, and release the drug to kill target cells. Therefore, when choosing which linker to be uses, the nature of the target should be considered. Selection of cytotoxic drugs Since the antibody enters the body and can effectively enter the tumor site by about 0.003–0.08% of the total amount, it is necessary to have a highly effective and highly sensitive killing effect on the target cells (free drug IC50: 10-1 1–10-9M). ). There are two main types of commonly used drugs at present - microtubule inhibitors and DNA-damaging agents. ADC development trend Site-specific conjugation At present, the most advanced ADC drugs are using traditional no-specific conjugation. The biggest disadvantage is that the product obtained is a mixture of different drug molecules per antibody. Site-specific conjugated drugs, and more importantly, uniform data (eg, PK) for clinical evaluation is difficult to obtain. In oder to solve these shortcomings, site specific conjugation technology has become a hot spot for major companies. Using site specific conjugation techniques, the same number of drug molecules can be carried on each antibody to obtain a uniform ADC drug. It is conducive to pharmacodynamic research and evaluation. And in the clinical can get more stable and effective results. Among them, A mbrx's Unatural Amino acid (pAcPhe) technology has more applications and promotion prospects. Multivalent ADC drugs The development of antibody drugs and vaccines has progressed from monovalent drugs to multivalent drugs. ADC might also follow this development process, that is, to link several small molecules that are synergistic with each other in the same antibody to improve the drug's efficacy. This requires a more sophisticated conjugation technology, to integrate two or more technologies. But now, the site-specific technology, excessively pursuits the coupling of a specific molecules at a specific site and neglects the diversity of coupling. Practical and traditional techniques for multivalent coupling of drugs require simultaneous coupling of multiple drugs on one antibody. In this case, the singularity of the antibody itself to modify the linking group will result in a mixed product, and there is no guarantee that each antibody carries a different drug at the same time. This problem can be solved by Site-specific techniques. When performing Site-specific modification, a variety of different coupling groups can be designed, which can use a group to carry out drug couples for the linker with the corresponding group. Monoclonal antibodies and their conjugates are macromolecular substances. Large drug molecules are difficult to penetrate deep into the solid tumor through the capillary endothelium and through the extracellular space of the tumor. The use of antibody fragments, such as Fab, to prepare conjugates with lower molecular weight, may increase the permeability to the extracellular space and increase the amount of drug reaching deep tumor cells. "Small size or moderate miniaturization is an important way to develop ADC drugs." Reference Beck, Alain, et al. "Strategies and challenges for the next generation of antibody–drug conjugates." Nature reviews Drug discovery 16.5 (2017): 315. Okeley NM, et al. (2010) Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin Cancer Res 16:888–897. Wu AM, Senter PD (2005) Arming antibodies: Prospects and challenges for immunoconjugates. Nat Biotechnol 23:1137–1146. Nagayama, Aiko, et al. "Antibody–Drug Conjugates for the Treatment of Solid Tumors: Clinical Experience and Latest Developments." Targeted oncology 12.6 (2017): 719-739. Polakis P (2005) Arming antibodies for cancer therapy. Curr Opin Pharmacol 5:382–387. Dan, Nirnoy, et al. "Antibody-drug conjugates for cancer therapy: chemistry to clinical implications." Pharmaceuticals 11.2 (2018): 32. Lambert JM (2005) Drug-conjugated monoclonal antibodies for the treatment of cancer. Curr Opin Pharmacol 5:543–549. Sau, Samaresh, et al. "Advances in antibody–drug conjugates: a new era of targeted cancer therapy." Drug discovery today (2017). Vater CA, Goldmacher VS (2010) Antibody-cytotoxic compound conjugates for oncology. Macromolecular Anticancer Therapeutics, Part 4:331–369. Zhou, Qun. "Site-specific antibody conjugation for ADC and beyond." Biomedicines 5.4 (2017): 64. Kovtun YV, Goldmacher VS (2007) Cell killing by antibody-drug conjugates. Cancer Lett 255:232–240.
  10. What is metabolomics? We know that metabolism is the general term for various chemical reactions in living organisms. It is one of the most important life activities of animals and plants. Individuals adapt to changes in internal and external environment through various metabolic adjustments, which is one of the basic characteristics of life activities. A "metabolome" is a collection of all metabolites in a biological sample. There are many kinds of metabolites (may be more than one million species) with various structures, and there are large differences in distribution and concentration in different tissues and body fluids. The main types include lipids, amino acids, organic acids, carbohydrates, nucleic acids, and so on. Metabolomics is an independent discipline, systematically studying metabolomes that provides unique biochemical fingerprints of all cellular processes. It can be used to identify metabolic biomarkers elucidating underlying disease mechanisms or predicting environmental changes or external interventions reaction. What is lipidomics? Lipolipomics is an independent discipline that systematically studies lipid groups. As a method for large-scale qualitative and quantitative study of lipid compounds and understanding their functions and changes under different physiological and pathological conditions, it can accurately and comprehensively provide a full-fat information spectrum of biological samples under different physiological conditions. As can be seen from the above definition, "lipolipomics" is actually a branch of "metabolomics." However, because lipid metabolism is the most major category of animal and plant metabolism (about 70% of metabolites in plasma is lipid), it is the hot spot of animal and plant metabolism research. Lipid metabolism is involved in energy transportation and intercellular information communication and network regulation in the process of growth and development. As a major component of cell membranes and lipid droplets, various structural lipids play an important role in a wide range of biological processes, such as signal transduction, transport, and biomacromolecule sorting with different biochemical properties. With the rapid development of lipidomics in recent years, scientists have gradually divided the lipid group research from mebabolism to “lipidomics”. Currently, when we refer to “metabolomics”, the lipid group analysis is no longer included. Clinical application of lipidomics studies Comprehensive analysis of lipids, i.e. lipidomics is a prerequisite for understanding the subtle dynamic changes and pathogenesis of lipids during cell changes. Lipid metabolism disorders and reproductive development defects are closely related to a variety of major diseases such as diabetes, cardiovascular disease, fatty liver, obesity, cancer, and Alzheimer's disease. The changes of lipid composition can directly reflect the physiological and pathological state of the organism. Therefore, it is of great significance to analyze the changes of lipid spectrometry during the growth and development of tissues and organs and to analyze the mechanism of related metabolic regulation network. With an integrated set of separation, characterization, identification and quantification system, featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective targeted lipidomics, untargeted lipidomics, Exosome Lipidomics and MALDI Imaging Lipidomics Service. Advantages of Creative Proteomics Services The compounds we test are widely covered, ranging from small water-soluble molecules to large lipids. We can analyze any biological materials, including but not limited to biofluids and tissues from animals, cell cultures and humans. A comprehensive platform contains advanced instruments, including MS, GC-MS, LC-MS, NMR, and so on. A complete analysis report is offered, including method interpretation, data, and result files.
  11. Lipids are the general term for fats, cholesterol, phospholipids, lipoproteins and glycolipids. It is one of the important nutrients needed by the human body. It provides the energy needed by the body and the essential fatty acids needed by the body. Lipid is one of the three nutrients that can produce energy, together with proteins and carbohydrates, and plays an important role in providing energy for the human body. It is the constituent of human cell tissue, among which phospholipids and cholesterol are the main components of all biofilms. The human body needs to absorb a certain amount of lipid substances every day, but excessive intake can lead to the occurrence and development of hyperlipidemia, atherosclerosis and other diseases. It is a compound insoluble in water but soluble in organic solvents, including fats and lipids. Fat accounts for about 15% of total human body weight, with a minimum of 13% and a maximum of 50%. Animal oils provide mainly saturated fatty acids while vegetable oils mainly unsaturated fatty acids. The main functions of lipids are: 1. Main components of cell membrane and biofilm, fixing body tissues and organs, and fat is the isolation layer of organs and joints, filling and avoiding friction. 2. Supply and store energy. Fat intake should be 20% to 25% of total energy. One gram of fat provides 37.6 kilojoules of heat. You also need 70 to 80 grams of fat per day. 3. Promote digestion and absorption of fat-soluble vitamins.4. Maintain body temperature. Here we will introduce some important lipids below: 1. Phospholipids Phospholipids are lipids containing phosphoric acid, which are composed of glycerophospholipids and sphingolipids. Glycerophospholipids are formed by condensation of phosphoric acid and glycerol, while sphingolipids are formed by condensation of sphingosine-1-phosphate and phosphoric acid. Phospholipids mainly act as emulsifiers, meaning that a molecule of phospholipids has both a hydrophilic part bound to water and a hydrophobic part bound to oil. So, it's an intermediate medium of water and oil that doesn't blend together, and makes it work very well. Most of the phospholipids in foods are in the form of soft phospholipids. As a plant extract, phospholipids are often found in egg yolks, animal livers and legumes. Egg yolk, soybean, fish head, sesame, mushroom, yam and black fungus also contain a certain number of phospholipids. 2. Sphingolipids Sphingolipids consist of a molecule of long-chain fatty acid, a molecule of sphingosine or its derivatives and a molecule of polar head alcohol. Sphingosine is the parent compound of many long chain amino alcohols in sphingolipids and is abundant in mammals. The polar head group of sphingolipids binds to the hydroxy group of sphingosines, while the fatty acid part forms amide bonds with the amino group. Therefore, sphingolipids, with a polar head group and two non-polar tails (long hydrocarbon chains of fatty acids and sphingolipids), are polar lipids, the second largest group of membrane lipids after phospholipids. Sphingolipids can be divided into sphingolipids, sphingolipids and gangliosides. 3. Steroids Steroids are a broad class of cyclopentane derivatives of total hydrogenation phenanthrene widely distributed in the biological world. Steroid compounds contain no bound fatty acids and are no saponifiable lipids; These compounds are isoprene like substances, which are generated by triterpene cyclization and chemical modification. Steroids include sterols (such as cholesterol, lanosterol, glusterol, soy sterol, ergosterol), bile acids and bile alcohols, steroid hormones (such as adrenocorticosteroids, androgen, estrogen), insect ecdysis hormones, cardiac glycosides, saponin ligands and bufotoxin. In addition, there are synthetic steroids such as anti-inflammatory agents (hydro prednisone, dexamethasone), steroids that promote protein synthesis and oral contraceptives. Anabolic steroids are similar to synthetic male hormones. They are a kind of synthetic chemical derivatives similar to human testosterone in structure and activity. On October 27, 2017, the world health organization's international agency for research on cancer released a preliminary list of carcinogens for reference, androgen (anabolic) steroids in the 2A list of carcinogens.
  12. Gene therapy: a genetically manipulated disease program Gene therapy refers to the introduction of normal genes into human cells to correct or supplement diseases caused by genetic defects or abnormalities. It is a fundamental therapeutic strategy. The introduced gene may be a homologous gene corresponding to the defective gene, expressing a specific function in vivo, or may be a therapeutic gene unrelated to the defective gene. In organisms, genetic information is transmitted step by step along the direction of "DNA-RNA-protein" (Central Dogma). Protein is a manifestation of genetic information, so the disease occurs mostly as a protein-level anomaly. At present, most of the drugs target proteins, such as small molecule targeted drugs for treating tumors and macromolecular monoclonal drugs, which are used to treat diseases by changing the function of proteins. However, Gene therapy starts from the upstream of the protein—DNA. It regulates DNA transmission to change the transmission of genetic information, thereby changing the properties of the protein and achieving disease treatment from the source. In addition, there are a small number of drugs, such as the small nucleic acid drug Patisiran, which target RNA. In a broad sense, these drugs are also in the category of gene therapy. In this article, we mainly talk about DNA-level. According to the central dogma of gene expression, each physiological process can be understood as the result of specific intensity expression of a particular gene in a specific time and space. If this balance is broken, it will induce disease. From this perspective, almost all diseases can be explained at the DNA level, which is the theoretical basis of gene therapy. According to the different types of gene mutations, the genetic abnormalities leading to disease can be roughly divided into two categories: (1). Gene mutations lead to protein dysfunction by gene-directed synthesis. T manifests as protein with no function, weakened function or over-function, or even produced harmful protein; (2). Abnormal gene expression, which is expressed by the expression of the gene that should not be expressed, the expression of the gene to be expressed, and the intensity of the gene expression being too high or too low. Therefore, in theory, gene therapy can cure most diseases. However, the occurrence of diseases often involves multiple genes, and the interaction between the corresponding proteins forms a huge regulatory network. It is difficult to achieve treatment for only one or several genes. In addition, scientists' research on human gene function and disease pathogenesis is still very limited, and there are a large number of undiscovered new genes and signal networks. Too many uncertainties in genes and diseases have greatly limited the application of gene therapy. Therefore, gene therapy is currently only applicable to a few diseases with very clear pathogenic mechanisms or treatment options, such as single-gene genetic diseases and tumors. Compared with conventional drugs/treatments, gene therapy can solve the disease from the source, so there are obvious advantages in some diseases that are currently untreated or poorly curable prognosis, such as hemophilia. In terms of safety, gene therapy still belongs to emerging technologies. There are still many blind spots in the understanding of genes and diseases, and it is usually difficult to reverse after genetic changes, and the potential risks are large. Most of the traditional drug treatments have gone through decades. Even after hundreds of years of development and use, the risks are relatively controllable. Therefore, we believe that conventional drugs are still the first choice for disease treatment in the future. Gene therapy is more like a supplement to conventional treatment options. The process of gene therapy According to different modes of administration, gene therapy can be divided into two categories: "in vivo" treatment and "in vitro" treatment. The "in vivo" gene therapy operation process is relatively simple, and can be roughly divided into three steps: (1) using a genetic engineering method to insert a normal gene into the DNA of a viral vector; (2) in vitro packaging of the recombinant viral DNA to produce fully engineered virus with infectious ability; (3) directly injecting the recombinant virus into the patient, the virus infects the diseased cells and brings the normal gene to the target cells to achieve treatment of the disease. The "in vitro" gene therapy can be divided into six steps: (1) inserting the normal gene into the DNA of the viral vector; (2) packaging the recombinant viral DNA in vitro to produce a fully engineered virus with infectious ability; (3) obtaining patient's somatic cells, such as hematopoietic stem cells, and undergo in vitro culture and amplification; (4) infecting the patient cells with the recombinant virus, and introducing the normal gene into the target cells; (5) in vitro, the recombinant cells carrying the normal gene The culture is expanded; (6) the recombinant cells carrying the normal gene are returned to the patient to realize the treatment of the disease.
  13. 8. PNAS: Scientists developed the first adult induced specific stem cells On April 4th, a research team from the University of New South Wales (UNSW) published an article in the journal PNAS, which was the first to develop a revolutionary stem cell repair technology. This technology can successfully re-inducing fat and bone cells into pluripotent stem cells, and is expected to be used in the treatment of human injuries, including spinal injuries and fractures. This technique is similar to the regeneration of Salamander limbs. Its most prominent achievement is the transformation of adult cells into induced immense stem cells (iMS cells), and iMS cells have the function of self-renewal and differentiation into many types of cells. The iMS cells can treat the human body damage caused by disease, aging or trauma, and will revolutionize the current state of regenerative medicine treatment of body damage. The first induced specific adult stem cell (iMS): capable of self-renewal repair, differentiation into multiple types of cells. According to the stage of development, stem cells can be divided into embryonic stem cells (ES cells, which have the ability to differentiate into intact individuals) and adult stem cells (somatic stem cells). Adult stem cells have the function of differentiating into specific cells and are not capable of differentiating into various cell types. The breakthrough in this latest technology published by PNAS is that induced specific stem cells can differentiate into many types of cells. 9. PNAS: adult adipocytes can differentiate into pluripotent stem cells, or can be used for tissue damage repair For the first time, Australian scientists have reprogrammed adult bones or fat cells to obtain stem cells that can differentiate into any tissue, thereby repairing damaged tissues and organs. Inspired by the phenomenon that lizards can regenerate limbs, researchers developed techniques that can return adult cells to the state of stem cells and gain the potential for division and multi-directional differentiation - pluripotent stem cells. This means that this part of the cell can repair damage to any part of the body: including the spinal cord, joints and muscles. The significance of this study is that there have been no previous reports of successful differentiation of adult stem cells into multiple types of tissues. "This technology is a revolutionary advancement in stem cell therapy, and there has never been evidence that adult stem cells can differentiate directly into tissues." John Pimanda, Principal Investigator from the University of New South Wales, said. 10. Nature: A major breakthrough! Human haploid embryonic stem cells was produced by the first time! Doi:10.1038/nature17408 In a study, researchers from the Hebrew University of Jerusalem, the University of Columbia Medical Center, and the New York Stem Cell Foundation Institute succeeded in producing a new type of embryonic stem cell that carries only a single copy of the human genome, rather than two copies of the human genome normally found in stem cells. The relevant research results were published online in the Nature Journal on March 16, 2016, and the title of the paper is "Derivation and differentiation of haploid human embryonic stem cells". The haploid embryonic stem cells described in this study are the first known human daughter cells capable of producing a single copy of the genome of a parental cell by cell division. Human cells are considered to be diploid because they inherit two sets of chromosomes, for a total of 46 chromosomes, 23 of which are from the mother and 23 from the father. The only exceptions are germ cells (eggs and sperm), which are haploid cells and contain a set of chromosomes, 23 chromosomes. These haploid cells cannot produce more eggs and sperm by dividing. Efforts to use human egg cells to produce embryonic stem cells have previously led to the production of diploid stem cells. In this study, the researchers promoted the division of unfertilized human eggs. They then labeled the DNA with a fluorescent dye to isolate these haploid embryonic stem cells, which were scattered among more diploid cells. With years of experience in providing high-quality products and services in the field of biopharmaceuticals for customers all over the world, scientists in Creative Biolabs will be more focused on developing first class product and technology to meet the need of stem cell therapy development. In cell therapy, iPS cell technology has opened huge possibilities for cell therapy and regenerative medicine. There are still significant challenges in gene and cell therapy, before these new approaches can enter main stream medicine. Significant safety issues have been reported in some trials and the need for better vectors, delivery techniques and treatment genes are widely recognized.
  14. 8. PNAS: Scientists developed the first adult induced specific stem cells On April 4th, a research team from the University of New South Wales (UNSW) published an article in the journal PNAS, which was the first to develop a revolutionary stem cell repair technology. This technology can successfully re-inducing fat and bone cells into pluripotent stem cells, and is expected to be used in the treatment of human injuries, including spinal injuries and fractures. This technique is similar to the regeneration of Salamander limbs. Its most prominent achievement is the transformation of adult cells into induced immense stem cells (iMS cells), and iMS cells have the function of self-renewal and differentiation into many types of cells. The iMS cells can treat the human body damage caused by disease, aging or trauma, and will revolutionize the current state of regenerative medicine treatment of body damage. The first induced specific adult stem cell (iMS): capable of self-renewal repair, differentiation into multiple types of cells. According to the stage of development, stem cells can be divided into embryonic stem cells (ES cells, which have the ability to differentiate into intact individuals) and adult stem cells (somatic stem cells). Adult stem cells have the function of differentiating into specific cells and are not capable of differentiating into various cell types. The breakthrough in this latest technology published by PNAS is that induced specific stem cells can differentiate into many types of cells. 9. PNAS: adult adipocytes can differentiate into pluripotent stem cells, or can be used for tissue damage repair For the first time, Australian scientists have reprogrammed adult bones or fat cells to obtain stem cells that can differentiate into any tissue, thereby repairing damaged tissues and organs. Inspired by the phenomenon that lizards can regenerate limbs, researchers developed techniques that can return adult cells to the state of stem cells and gain the potential for division and multi-directional differentiation - pluripotent stem cells. This means that this part of the cell can repair damage to any part of the body: including the spinal cord, joints and muscles. The significance of this study is that there have been no previous reports of successful differentiation of adult stem cells into multiple types of tissues. "This technology is a revolutionary advancement in stem cell therapy, and there has never been evidence that adult stem cells can differentiate directly into tissues." John Pimanda, Principal Investigator from the University of New South Wales, said. 10. Nature: A major breakthrough! Human haploid embryonic stem cells was produced by the first time! Doi:10.1038/nature17408 In a study, researchers from the Hebrew University of Jerusalem, the University of Columbia Medical Center, and the New York Stem Cell Foundation Institute succeeded in producing a new type of embryonic stem cell that carries only a single copy of the human genome, rather than two copies of the human genome normally found in stem cells. The relevant research results were published online in the Nature Journal on March 16, 2016, and the title of the paper is "Derivation and differentiation of haploid human embryonic stem cells". The haploid embryonic stem cells described in this study are the first known human daughter cells capable of producing a single copy of the genome of a parental cell by cell division. Human cells are considered to be diploid because they inherit two sets of chromosomes, for a total of 46 chromosomes, 23 of which are from the mother and 23 from the father. The only exceptions are germ cells (eggs and sperm), which are haploid cells and contain a set of chromosomes, 23 chromosomes. These haploid cells cannot produce more eggs and sperm by dividing. Efforts to use human egg cells to produce embryonic stem cells have previously led to the production of diploid stem cells. In this study, the researchers promoted the division of unfertilized human eggs. They then labeled the DNA with a fluorescent dye to isolate these haploid embryonic stem cells, which were scattered among more diploid cells. With years of experience in providing high-quality products and services in the field of biopharmaceuticals for customers all over the world, scientists in Creative Biolabs will be more focused on developing first class product and technology to meet the need of stem cell therapy development. In cell therapy, iPS cell technology has opened huge possibilities for cell therapy and regenerative medicine. There are still significant challenges in gene and cell therapy, before these new approaches can enter main stream medicine. Significant safety issues have been reported in some trials and the need for better vectors, delivery techniques and treatment genes are widely recognized.
  15. Abstract: Cellular function assay has irreplaceable significance and value for today's clinical medicine. Immunocyte function tests include in vivo and in vitro assay. The in vitro test of immune cellular function was mainly designed according to the characteristics of proliferation activity and secretion killing activity of immune cells. Specific immune cellular function tests include: 1. Lymphocyte T cell function test, including T cellular proliferation test, T cellular secretion function test, T cellular mediated cytotoxicity test and in vivo test; lymphocyte B cellular proliferation test, hemolytic plaque test, enzyme-linked immunospot assay and in vivo assay. The natural killer cellular activity assay methods include: enzyme release method, radionuclide release method, chemiluminescence method, flow cytometry method, and the like. 2. Phagocytic function test, which further includes chemotactic function detection, phagocytosis and bactericidal function assay. In addition, the detection of islet β-cellular function is an important component of the pathophysiological study of abnormal glucose metabolism. It is clear that the islet β-cellular function status plays an important role in guiding clinically effective interventions for abnormal glucose metabolism. This article will discuss the process and significance of the above-mentioned cellular function assays. Keywords: lymphocytes, phagocytic cellular, functional assay Assay of lymphocyte function What is a lymphocyte? Lymphocytes are a type of white blood cells, which are the smallest white blood cells produced by lymphoid organs and are important cellular components of the body's immune response function. Lymphocytes are a kind of cellular line with immune recognition function. According to their migration, surface molecules and functions, lymphocytes can be divided into T lymphocytes, B lymphocytes and natural killer cells. The results and significance of lymphocyte functional assay Here, we describe the value of lymphocyte function assays in a disease called henoch-Schonlein purpura (HSP). HSP is the most common autoimmune response mediated systemic vasculitis in childhood. At present, its pathogenesis has not been fully elucidated. In recent years, studies have found that there are immune dysfunctions in the body of patients with HSP. Bacteria or infections and food allergies are the main causes. Cellular lymphatic function assay found that NK cells are a group of non-specific immune effector cells, which can not only directly kill viruses and target cells but also have immunoregulatory functions. Besides, NK cells can directly recognize B lymphocytes and affect the differentiation and proliferation of B lymphocytes. When the activity of NK cells is decreased, the body's inhibition of differentiation and proliferation of B lymphocytes is weakened, and the activity of B lymphocytes is increased. The body's humoral immune function leads to increased secretion of Ig. The synthetic antigen-antibody complex increases and deposits on the blood vessel wall and glomerular basement membrane, activating complement and causing damage to vascular endothelial cellular and basement membrane. At the same time, excessive secretion of inflammatory mediators, acting on small blood vessel smooth muscles, expansion of small arteries and capillaries, increased permeability, causing edema and bleeding of skin, mucous membranes and internal organs. This is the pathogenesis of HSP. In summary, children with HSP have cellular and humoral immune dysfunction in the acute phase, and peripheral blood lymphocyte function is important for the early diagnosis of this disease. Assay of phagocytic function Measuring test process It has been shown that intraperitoneal injection of sodium thioglycolate in mice stimulates aggregation of macrophages. Four days later, the mice were injected with a suspension of sheep red blood cells in the peritoneal cavity. After one hour, the peritoneal phagocytic cells were dissected and collected. The phagocytosis of sheep red blood cells was observed by staining and microscopic examination. The phagocytic function of phagocytic cells can be determined by calculating the percentage of phagocytosis or the phagocytic index. Results andevaluation Studies have shown that in severe liver disease, the mononuclear macrophage function in vivo, including cellular adhesion chemical tendency, phagocytic bacteria and killing effects, and the ability to release various alcohols are inhibited, may be related to the presence of inhibitors in the body. It has nothing to do with the cause of liver disease. Once the liver disease recovers, its phagocytic function is also normal. Under normal conditions or in chronic liver injury, dead Kupffer cells are supplemented by proliferating division of adjacent cells. In acute liver injury, Kupffer cells are supplemented by circulating macrophages from the myeloproliferative surface. Islet beta cell function assay What is the islet β cell function? Islet beta cell function refers to the pulse-like secretion of insulin and the ability to release or secrete insulin and stimulate other peptides caused by various stimuli. There are many methods for detecting islet beta cell function, including detection of insulin pulse-like secretion patterns and islet beta cell secretion stimulation tests (glucose stimulation test and non-glucose stimulation test). Glucose stimulation test mainly includes high glucose clamp technique, intravenous glucose tolerance test (IVGTT) combined with mathematical model analysis technology and clinically commonly used oral glucose tolerance test (OGTT), while non-sugar stimulation test mainly includes arginine test and glucagon Prime test. In addition, it is also possible to judge its function by analyzing other peptides secreted by islet β cells, such as C peptide, proinsulin, amylin, and the like. A plurality of indicators can be obtained by the above detection method, and the islet β cellfunction is quantitatively evaluated. The choice of islet beta cell function detection methods Islet beta cell dysfunction in the process of diabetes is multi-faceted. Insulin pulse-like secretion mode, PI level, and detection of insulin-phase secretion reaction after glucose stimulation can be performed in normal glucose tolerance populations with high risk factors for diabetes, who might be first-degree relatives of elderly individuals and diabetic patients. The insulin phase 1 secretory response of the arginine test may be abnormal after the onset of diabetes, and the glucagon test may determine the islet β cell dysfunction at a later stage. Therefore, appropriate functional tests can be selected for the purpose of the study. In the normal sugar regulation stage, high glucose clamps can be used to understand the possible defects of islet β cells in high-risk populations; In the process of gradual decline of islet β cells, IVGTT and OGTT can be used to evaluate insulin secretion indicators early; After the occurrence of diabetes, the arginine test and the change of the 2-phase insulin secretion reaction of OGTT can be used as indicators to judge the severity of the disease; When the islet β cell function is about to fail,only the glucagon test can be used to judge the degree of exhaustion. In summary, the selection of islet beta cell function testing methods depends on the purpose of the test and is considered in combination with the stage in which the individual is in the natural course of diabetes. If you want to find potential islet β-cell defects, especially in evaluating drug efficacy and research mechanism, you should choose methods that are more accurate and sensitive. When conducting large-scale clinical research, the method to be chosen should be convenient and reproducible. A method for detecting islet β-cell function often fails to fully reflect the function state of islet β-cell, so it is often necessary to use a combination of methods to judge comprehensively. Conclusion In the prevention and treatment of diabetes, simulating or restoring the phase secretion and pulse secretion of insulin and effectively improving insulin sensitivity will achieve good therapeutic effects. Appropriate functional tests based on the characteristics of different individuals can help to accurately evaluate the islet β-cell functional status and insulin resistance, which is of great significance for the development of individualized treatment plans and scientific judgment of prognosis from the perspective of pathophysiology. References [1] Liu X, Li YR, Hu LH, et al. High frequencies of HLAB27 in Chinese patients with suspected of ankylosing spondylitis [J]. Rheumatol Int, 2010, 30(10):1305-1308